Traumatic disruption of bone and the interruption of blood supply to bone initiate a cascade of events eventually leading to full repair of bone given the circumstances that mesenchymal stem cells (MSCs) can differentiate into osteoblasts (OBs) and that new blood supply can be re-established. A key step of bone repair occurs in the early inflammatory phase where there is an elaboration of CXC chemokines. CXC chemokines having a signature glu-leu-arg (ELR) sequence upstream to the CXC motif (ELR+ CXC chemokines) are also uniquely angiogenic. These ELR+ CXC chemokines bind to either CXC receptor 1 (CXCR1) and/or CXC receptor 2 (CXCR2), to enable their chemotactic and angiogenic effects. It is hypothesized that the ELR+ CXC chemokines, CXCL5 and CXCL8, elaborated during the inflammatory phase, play a key role in intramembranous bone repair by 1) stimulating chemotaxis of MSCs to achieve a critical mass of MSCs at the repair site, 2) aiding in the differentiation of MSCs into OBs during bone repair, and 3) initiating angiogenesis to support MSC condensation and granulation tissue in the inflammatory phase of osteogenic healing. There is evidence that canonical Wnt/2-catenin signaling is required for new bone formation. Thus we further hypothesize that canonical Wnt/2-catenin signaling is mechanistically involved in the elaboration of CXCL5 and CXCL8 with subsequent paracrine stimulation of angiogenesis, and that CXCL5 and CXCL8 can also foster MSC migration and osteogenic differentiation via the upregulation of protein kinase D (PKD) and activation of the mitogen-activated protein kinase (MAPK) signaling pathways to enable bone repair. The specific aims are to: 1) demonstrate that the elaboration of CXCL5 and CXCL8 during the initiation of osteogenic differentiation occurs through the Wnt/2-catenin signaling pathway; 2) show that CXCL5 and CXCL8 through CXCR1 and/or CXCR2 leads to migration and osteogenic differentiation of human and mouse MSCs (hMSC; mMSC) models in vitro and that cellular signaling leading to migration and osteogenic differentiation occurs through PKD and downstream activation of the MAPK pathways; 3) establish if there is a mMSC (from bone marrow) or OB- specific defect in migration and differentiation due to the lack of the mouse CXC receptor (mCXCR) (homologous to human CXCR2), and/or if the bone phenotype and less bone healing found in mCXCR null mice is due to insufficient ELR+ CXC chemokine-stimulated angiogenesis. ELR+ CXC and osteogenic markers mRNA and protein will be analyzed by real-time RT-PCR and ELISA, respectively. MSC migration will be done by Transwell assay. Overexpression or inhibition of CXC receptors and downstream PKD and MAPK signaling will be done using transfected expression vectors or siRNA technology, respectively. In vivo studies will use histomorphometric analyses to assess bone healing and new blood vessel formation in a calvarial defect model in mCXCR knockout mice. Understanding the early events in bone repair may help to develop and deliver therapies that would hasten bone healing to restore bone to pre-morbid levels of strength and soundness. PUBLIC HEALTH RELEVANCE: RELEVANCE TO VETERANS HEALTH The current research proposal is every relevant to the care of veterans who have experienced traumatic injury in active combat theaters. Characterization of the types of combat injuries observed in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) has shown that traumatic bone injury is a prevalent problem. In fact the types of combat injuries observed in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) has shown that 26% of all extremity wounds are fractures (Owens BD, Kragh, Jr JF, Macaitis J, Svoboda SJ, Wenke JC. Characterization of extremity wounds in Operation Iraqi Freedom and Operation Enduring Freedom. J Orthop Trauma 21:254-257, 2007). Head and face trauma accounts for 2% - 20% of combat injuries depending on the theater of Middle East conflict. Thus traumatic bone injury is found to be associated with active military campaigns. In those traumatic bone injuries that either heal slowly or do not heal, or in the case of facial and skull bone injuries that could also have attendant visual or hearing deficits or traumatic brain injury, increased morbidity, long-term rehabilitation, and even long-term disabilities are faced by such veterans that take a toll on the veterans themselves, their families, and the financial resources of the country. It is important to understand the early events in bone repair. These events include the generation of new bone forming cells called osteoblasts from more primitive cells from bone marrow (e.g. mesenchymal stem cells), and the restoration of new blood supply to bone. Re-establishing blood supply to bone by a process called angiogenesis (the sprouting of new blood vessels from existing blood vessels after injury) is necessary and critical for bone repair to occur. Knowledge of these key elements in bone repair may help to develop and deliver therapies that would hasten bone healing to restore bone to pre-morbid levels of strength and soundness.